Antisense modulation of Interferon gamma receptor 1 expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of Interferon gamma receptor 1. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding Interferon gamma receptor 1. Methods of using these compounds for modulation of Interferon gamma receptor 1 expression and for treatment of diseases associated with expression of Interferon gamma receptor 1 are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of Interferon gamma receptor 1. In particular, thisinvention relates to compounds, particularly oligonucleotides,specifically hybridizable with nucleic acids encoding Interferon gammareceptor 1. Such compounds have been shown to modulate the expression ofInterferon gamma receptor 1.

BACKGROUND OF THE INVENTION

Cytokines are small to medium sized proteins and glycoproteins thatmediate highly potent biological effects on many cell types in networksor cascades. They have critical roles in hematopoiesis, inflammatoryresponses and the development and maintenance of immune responses.Typical properties in networks of cytokines are pleiotropy, redundancy,synergistic activity and antagonistic effects upon each other (Townsendand McKenzie, J. Cell Sci., 2000, 113, 3549-3550). Knowledge of howcytokine networks are comprised and operate is important inunderstanding how they mediate their diverse effects on biologicalsystems (Townsend and McKenzie, J. Cell Sci., 2000, 113, 3549-3550).

Interferon gamma (IFN-gamma) is a cytokine secreted by activated Tlymphocytes and natural killer (NK) cells. It is involved in themodulation of a wide variety of immunological and inflammatory responsesincluding activation of macrophages, cytotoxic T cells and NaturalKiller (NK) cells, regulation of antibody production by B lymphocytesand control of apoptosis (Billiau, Adv. Immunol., 1996, 62, 61-130).

Interferon gamma receptor complexes are expressed on almost allnucleated cells and show species specificity in their ability to bindinterferon gamma (Dorman and Holland, Cytokine Growth Factor Rev., 2000,11, 321-333). The functional interferon gamma receptor is composed oftwo 90 kDa polypeptides designated interferon gamma receptor 1 (alsoknown as IFNGR1, IFN-gamma receptor-alpha chain and CD119w) andinterferon gamma receptor 2 (also known as IFNGR2, IFN-gammareceptor-beta, IFN-gamma transducer 1, AF-1 and GAF).

The extracellular portion of interferon gamma receptor 1 contains theIFN-gamma ligand-binding domain and the intracellular portion containsthe domains necessary for signal transduction and receptor recycling(Dorman and Holland, Cytokine Growth Factor Rev., 2000, 11, 321-333). Inthe absence of stimulation, interferon gamma receptors 1 and 2 are notstrongly associated with each other. Upon binding of IFN-gamma as ahomodimer to the two interferon gamma receptor 1 proteins, interferongamma receptors 1 and 2, and their constitutively-associated Januskinases (JAK1 and JAK2, respectively) are brought into proximity so thatthe signaling cascade can begin via phosphorylation of tyrosine-440 oninterferon gamma receptor 1 (Dorman and Holland, Cytokine Growth FactorRev., 2000, 11, 321-333). Signals are transmitted to the nucleus via theJak-STAT mechanism of signal transduction involving a cascade oftyrosine phosphorylations of STAT proteins (signal transducer andactivator of transcription) in the cytoplasm.

Human interferon gamma receptor 1 has been mapped to chromosome6q23-6q24, a region which undergoes rearrangements in ovarian carcinomaand malignant melanoma (Le Coniat et al., Hum. Genet., 1989, 84, 92-94).In addition, partial deletions and translocations involving bands 6q14to 6q27 are frequently observed in various types of lymphoid cellmalignancies, leukemias and lymphomas (Le Coniat et al., Hum. Genet.,1989, 84, 92-94).

Complete absence of human IFN-gamma responsiveness due to a mutation ineither interferon gamma receptor 1 or 2 is typically associated withsevere mycobacterial infections in lungs, viscera, lymph nodes, bloodand bone marrow (Dorman and Holland, Cytokine Growth Factor Rev., 2000,11, 321-333). A clinical phenotype associated with autosomal dominantmutations of interferon gamma receptor 1 (leading to partial interferongamma receptor 1 deficiency) is milder than seen in individuals withcomplete absence of INF-gamma responsiveness and infections are usuallyresponsive to appropriate antimicrobial therapy (Dorman and Holland,Cytokine Growth Factor Rev., 2000, 11, 321-333.).

The expression of the human interferon gamma receptor complex ismodulated by environmental signals in human malignant T cells andthymocytes (Novelli et al., J. Immunol., 1994, 152, 496-504). Theupregulation of IFN-gamma expression promotes T cell proliferation in Tcells with low levels of interferon gamma receptor complex and apoptosisin T cells with high levels of interferon gamma receptor complex(Novelli et al., J. Immunol., 1994, 152, 496-504).

Although IFN-gamma signaling typically exerts antiviral effects, in somecases of bacterial infections, endogenous IFN-gamma can act as adetriment to the host. In the case of HIV infection, activation ofmonocytoid cells by IFN-gamma signaling was found to stimulate ratherthan inhibit virus replication (Biswas et al., J. Exp. Med., 1992, 176,739-750).

In addition, there are conflicting reports on the effects of IFN-gammasignaling on tumor growth in mice. For example, investigators haveinserted the IFN-gamma gene into non-immunogenic murine tumor cells withhigh metastasizing potential and found that the IFN-gamma secretingcells had less ability to develop tumors than the parental cells(Gansbacher et al., Cancer Res., 1990, 50, 7820-7825). On the otherhand, a metastasizing murine mammary carcinoma productively transfectedwith the IFN-gamma gene was found to metastasize more extensively thanthe untransfected tumor line (Ferrantini et al., J. Immunol., 1994, 153,4604-4615).

In experimental animal models of autoimmune disease including:autoimmune thyroiditis, experimental autoimmune peripheral neuritis andautoimmune insulin-dependent diabetes, experimental autoimmuneencephalomyelitis, autoimmune arthritis and autoimmune insulinitis,IFN-gamma was either found to facilitate induction of disease or toaggravate disease manifestations (Billiau, Adv. Immunol., 1996, 62,61-130). Increased levels of IFN-gamma mRNA have been observed inintestinal propria lamina of Crohn's disease patients (Fais et al., J.Interferon Res., 1994, 14, 235-238).

Deregulation of IFN-gamma signaling has been implicated in autoimmunedisease, complications due to viral infections and cancer. Furthermore,deregulated apoptosis signaling may impinge on other age-relateddisorders such as osteoporosis and atherosclerosis. Modulation ofexpression of components of the interferon gamma receptor complex,including interferon gamma receptor 1 may prove to be a useful strategywith which to down-regulate IFN-gamma signaling in cases where excessivesignaling precipitates disease.

Attempts to modulate the process of IFN-gamma signaling have thus farbeen essentially limited to inhibition of IFN-gamma itself. Strategiesaimed at inhibition of interferon gamma receptor 1 function are as yetuntested as investigative or therapeutic protocols. Consequently thereremains a long felt need for agents capable of effectively inhibitinginterferon gamma receptor 1 function.

Antisense technology is emerging as an effective means for reducing theexpression of specific gene products and may therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications for the modulation of interferon gamma receptor 1expression.

The present invention provides compositions and methods for modulatinginterferon gamma receptor 1 expression.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, particularly antisenseoligonucleotides, which are targeted to a nucleic acid encodingInterferon gamma receptor 1, and which modulate the expression ofInterferon gamma receptor 1. Pharmaceutical and other compositionscomprising the compounds of the invention are also provided. Furtherprovided are methods of modulating the expression of Interferon gammareceptor 1 in cells or tissues comprising contacting said cells ortissues with one or more of the antisense compounds or compositions ofthe invention. Further provided are methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of Interferon gamma receptor 1 byadministering a therapeutically or prophylactically effective amount ofone or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding Interferon gamma receptor 1, ultimatelymodulating the amount of Interferon gamma receptor 1 produced. This isaccomplished by providing antisense compounds which specificallyhybridize with one or more nucleic acids encoding Interferon gammareceptor 1. As used herein, the terms “target nucleic acid” and “nucleicacid encoding Interferon gamma receptor 1” encompass DNA encodingInterferon gamma receptor 1, RNA (including pre-mRNA and mRNA)transcribed from such DNA, and also cDNA derived from such RNA. Thespecific hybridization of an oligomeric compound with its target nucleicacid interferes with the normal function of the nucleic acid. Thismodulation of function of a target nucleic acid by compounds whichspecifically hybridize to it is generally referred to as “antisense”.The functions of DNA to be interfered with include replication andtranscription. The functions of RNA to be interfered with include allvital functions such as, for example, translocation of the RNA to thesite of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity which may be engaged in or facilitated by the RNA. The overalleffect of such interference with target nucleic acid function ismodulation of the expression of Interferon gamma receptor 1. In thecontext of the present invention, “modulation” means either an increase(stimulation) or a decrease (inhibition) in the expression of a gene. Inthe context of the present invention, inhibition is the preferred formof modulation of gene expression and mRNA is a preferred target.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding Interferon gamma receptor 1. The targeting processalso includes determination of a site or sites within this gene for theantisense interaction to occur such that the desired effect, e.g.,detection or modulation of expression of the protein, will result.Within the context of the present invention, a preferred intragenic siteis the region encompassing the translation initiation or terminationcodon of the open reading frame (ORF) of the gene. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon”. A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding Interferon gamma receptor 1, regardless of thesequence(s) of such codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5′ untranslatedregion (5′UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′—5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense and other compounds of the invention which hybridize to thetarget and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds which hybridize to these active sites.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

For use in kits and diagnostics, the antisense compounds of the presentinvention, either alone or in combination with other antisense compoundsor therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

Expression patterns within cells or tissues treated with one or moreantisense compounds are compared to control cells or tissues not treatedwith antisense compounds and the patterns produced are analyzed fordifferential levels of gene expression as they pertain, for example, todisease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds which affect expressionpatterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SURF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat oligonucleotides can be useful therapeutic modalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. This term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentinternucleoside (backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 50 nucleobases (i.e.from about 8 to about 50 linked nucleosides). Particularly preferredantisense compounds are antisense oligonucleotides, even more preferablythose comprising from about 12 to about 30 nucleobases. Antisensecompounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and borano-phosphateshaving normal 3′—5′ linkages, 2′—5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be abasic (the nucleobase is missing or has a hydroxylgroup in place thereof). Various salts, mixed salts and free acid formsare also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

A further prefered modification includes Locked Nucleic Acids (LNAs) inwhich the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of thesugar ring thereby forming a bicyclic sugar moiety. The linkage ispreferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof aredescribed in WO 98/39352 and WO 99/14226.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. No. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (−C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. No. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. The compounds of the invention caninclude conjugate groups covalently bound to functional groups such asprimary or secondary hydroxyl groups. Conjugate groups of the inventioninclude intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of Interferon gamma receptor 1 is treated by administeringantisense compounds in accordance with this invention. The compounds ofthe invention can be utilized in pharmaceutical compositions by addingan effective amount of an antisense compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the antisensecompounds and methods of the invention may also be usefulprophylactically, e.g., to prevent or delay infection, inflammation ortumor formation, for example.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingInterferon gamma receptor 1, enabling sandwich and other assays toeasily be constructed to exploit this fact. Hybridization of theantisense oligonucleotides of the invention with a nucleic acid encodingInterferon gamma receptor 1 can be detected by means known in the art.Such means may include conjugation of an enzyme to the oligonucleotide,radiolabelling of the oligonucleotide or any other suitable detectionmeans. Kits using such detection means for detecting the level ofInterferon gamma receptor 1 in a sample may also be prepared.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Preferredlipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidylglycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively,oligonucleotides may be complexed to lipids, in particular to cationiclipids. Preferred fatty acids and esters include but are not limitedarachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylicacid, capric acid, myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine,an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM),monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.Topical formulations are described in detail in U.S. patent applicationSer. No. 09/315,298 filed on May 20, 1999 which is incorporated hereinby reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Prefered bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Preferedfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also prefered are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly prefered combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999) each of which is incorporated herein by referencein their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other. In general, emulsions may be eitherwater-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueousphase is finely divided into and dispersed as minute droplets into abulk oily phase the resulting composition is called a water-in-oil (w/o)emulsion. Alternatively, when an oily phase is finely divided into anddispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil-in-water (o/w) emulsion. Emulsions maycontain additional components in addition to the dispersed phases andthe active drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p.92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited todaunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Numerous examples of antisensecompounds are known in the art. Two or more combined compounds may beused together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-alkoxy amidites

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham Mass. or GlenResearch, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleosideamidites are prepared as described in U.S. Pat. No. 5,506,351, hereinincorporated by reference. For oligonucleotides synthesized using2′-alkoxy amidites, the standard cycle for unmodified oligonucleotideswas utilized, except the wait step after pulse delivery of tetrazole andbase was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2′-Fluoro amidites

2′-Fluorodeoxyadenosine amidites

2′-fluoro oligonucleotides were synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyrylarabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

2′-Fluorodeoxycytidine

2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-O-(2-Methoxyethyl) Modified amidites

2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.).

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by TLC by first quenching the TLC sample with the addition ofMeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3′-O-Acetyl-2′-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH40H (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, TLC showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) wereadded with stirring, under a nitrogen atmosphere. The resulting mixturewas stirred for 20 hours at room temperature (TLC showed the reaction tobe 95% complete). The reaction mixture was extracted with saturatedNaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes wereback-extracted with CH₂Cl₂ (300 mL), and the extracts were combined,dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the artas 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×L) andbrine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to −10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 mL) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O -([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.639, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) nucleoside amidites

2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog may be obtained by selective2′-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minoramount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine ribosidemay be resolved and converted to 2′-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection proceduresshould afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosineand2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may bedisplaced by N-hydroxyphthalimide via a Mitsunobu reaction, and theprotected nucleoside may phosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the artas 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleosideamidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowlyadded to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyluridine

To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2 Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,46.9,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3 Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethyl-hydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]—[2′-deoxy]—[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2′ positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to ½ volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxy-ethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxyPhosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxyphosphorothioate]—[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toUnited States patent 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing 4 cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, Ribonucleaseprotection assays, or RT-PCR.

T-24 Cells

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

NHDF Cells

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

Treatment with Antisense Compounds

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2¹-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10 Analysis of Oligonucleotide Inhibition of Interferon GammaReceptor 1 Expression

Antisense modulation of Interferon gamma receptor 1 expression can beassayed in a variety of ways known in the art. For example, Interferongamma receptor 1 mRNA levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), or real-time PCR(RT-PCR). Real-time quantitative PCR is presently preferred. RNAanalysis can be performed on total cellular RNA or poly(A)+mRNA. Methodsof RNA isolation are taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis isroutine in the art and is taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, JohnWiley & Sons, Inc., 1996. Real-time quantitative (PCR) can beconveniently accomplished using the commercially available ABI PRISM™7700 Sequence Detection System, available from PE-Applied Biosystems,Foster City, Calif. and used according to manufacturer's instructions.

Protein levels of Interferon gamma receptor 1 can be quantitated in avariety of ways well known in the art, such as immunoprecipitation,Western blot analysis (immunoblotting), ELISA or fluorescence-activatedcell sorting (FACS). Antibodies directed to Interferon gamma receptor 1can be identified and obtained from a variety of sources, such as theMSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), orcan be prepared via conventional antibody generation methods. Methodsfor preparation of polyclonal antisera are taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2,pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation ofmonoclonal antibodies is taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5,John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11 Poly(A)+mRNA Isolation

Poly(A)+mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC™ manifold and blotted dry oh paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-time Quantitative PCR Analysis of Interferon GammaReceptor 1 mRNA Levels

Quantitation of Interferon gamma receptor 1 mRNA levels was determinedby real-time quantitative PCR using the ABI PRISM™ 7700 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR, in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., JOE, FAM, or VIC, obtained from either OperonTechnologies Inc., Alameda, Calif. or PE-Applied Biosystems, FosterCity, Calif.) is attached to the 5′ end of the probe and a quencher dye(e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda,Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the3′ end of the probe. When the probe and dyes are intact, reporter dyeemission is quenched by the proximity of the 3′ quencher dye. Duringamplification, annealing of the probe to the target sequence creates asubstrate that can be cleaved by the 5′-exonuclease activity of Taqpolymerase. During the extension phase of the PCR amplification cycle,cleavage of the probe by Taq polymerase releases the reporter dye fromthe remainder of the probe (and hence from the quencher moiety) and asequence-specific fluorescent signal is generated. With each cycle,additional reporter dye molecules are cleaved from their respectiveprobes, and the fluorescence intensity is monitored at regular intervalsby laser optics built into the ABI PRISM™ 7700 Sequence DetectionSystem. In each assay, a series of parallel reactions containing serialdilutions of mRNA from untreated control samples generates a standardcurve that is used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLtotal RNA solution. The RT reaction was carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent from Molecular Probes. Methods of RNA quantification byRiboGreen™ are taught in Jones, L. J., et al, Analytical Biochemistry,1998, 265, 368-374.

In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen reagentdiluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 25 uL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nmand emission at 520 nm.

Probes and primers to human Interferon gamma receptor 1 were designed tohybridize to a human Interferon gamma receptor 1 sequence, usingpublished sequence information (GenBank accession number NM_(—)000416,incorporated herein as SEQ ID NO:3). For human Interferon gamma receptor1 the PCR primers were:

forward primer: CTTAGAAAAGGAGGTGGTCTGTGAA (SEQ ID NO: 4) reverse primer:CCTGGATTGTCTTCGGTATGC (SEQ ID NO: 5) and the PCR probe was:FAM-CGTTGTCTCCAGCAACAGTTCCAGGC-TAMRA (SEQ ID NO: 6) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye. For human GAPDH the PCR primers were:

forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7) reverse primer:GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR probe was: 5′JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) where JOE (PE-AppliedBiosystems, Foster City, Calif.) is the fluorescent reporter dye) andTAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 14 Northern Blot Analysis of Interferon Gamma Receptor 1 mRNALevels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human Interferon gamma receptor 1, a human Interferon gammareceptor 1 specific probe was prepared by PCR using the forward primerCTTAGAAAAGGAGGTGGTCTGTGAA (SEQ ID NO: 4) and the reverse primerCCTGGATTGTCTTCGGTATGC (SEQ ID NO: 5). To normalize for variations inloading and transfer efficiency membranes were stripped and probed forhuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Inhibition of Human Interferon Gamma Receptor 1Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human Interferon gammareceptor 1 RNA, using published sequences (GenBank accession numberNM_(—)000416, incorporated herein as SEQ ID NO: 3, and the complement ofresidues 59001-85000 of GenBank accession number AL050337 which isincorporated herein as SEQ ID NO: 10). The oligonucleotides are shown inTable 1. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the oligonucleotide binds.All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout th e oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human Interferon gamma receptor 1 mRNA levels byquantitative real-time PCR as described in other examples herein. Dataare averages from two experiments. If present, “N.D.” indicates “nodata”.

TABLE 1 Inhibition of human Interferon gamma receptor 1 mRNA levels bychimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap TARGET TARGET % SEQ ISIS # REGION SEQ ID NO SITE SEQUENCEINHIB ID NO 133747 Coding 3 186 tggcatgatctggtactccc 95 11 133782 Coding3 1120 ccacgtcaggaatattttct 79 12 133783 Coding 3 1156tctctctctctattggagtc 69 13 133785 Coding 3 1204 aagcgatgctgccaggttca 014 133794 Coding 3 1490 gaaaattctttggaatcttc 0 15 133795 Stop Codon 31502 agctgatctcatgaaaattc 79 16 133799 3′ UTR 3 1572tcataatcttttcatgaaat 60 17 133800 3′ UTR 3 1579 tctgagatcataatcttttc 9218 133802 3′ UTR 3 1642 cctctttacgctttcatgta 4 19 133818 Intron 2 1015268 ccctgccccagagatttgtg 11 20 147599 Coding 3 89 gcggtgcccatctcagccct1 21 147600 Coding 3 98 cccagatccgcggtgcccat 92 22 147601 Coding 3 125ttagttggtgtaggcactga 0 23 147602 Coding 3 128 acattagttggtgtaggcac 84 24147603 Coding 3 148 tgttataggattcaattgta 70 25 147604 Coding 3 163atacgatagggttcatgtta 0 26 147605 Coding 3 174 gtactcccaatatacgatag 14 27147606 Coding 3 177 ctggtactcccaatatacga 85 28 147607 Coding 3 180gatctggtactcccaatata 77 29 147608 Coding 3 193 ggacctgtggcatgatctgg 7930 147609 Coding 3 306 tggatcaccaacatgatcag 0 31 147610 Coding 3 340ccctggctttaactctgacc 0 32 147611 Coding 3 344 ccaaccctggctttaactct 1 33147612 Coding 3 347 tgtccaaccctggctttaac 40 34 147613 Coding 3 370actttgcataggcagattct 0 35 147614 Coding 3 373 ctgactttgcataggcagat 0 36147615 Coding 3 387 tacagcaaattcttctgact 9 37 147616 Coding 3 420cagtttaggtggtccaattt 14 38 147617 Coding 3 428 ctgatatccagtttaggtgg 0 39147618 Coding 3 450 catgatttgcttctcctcct 0 40 147619 Coding 3 489gtctccatttacaaaaactg 67 41 147620 Coding 3 498 ttcctgctcgtctccattta 0 42147621 Coding 3 505 aatcgacttcctgctcgtct 0 43 147622 Coding 3 530atgtaacaggtagtttcggg 65 44 147623 Coding 3 554 ctcacatacacattgtacac 6945 147624 Coding 3 559 tcattctcacatacacattg 11 46 147625 Coding 3 565ttccgttcattctcacatac 0 47 147626 Coding 3 592 gcgtgagtattttatactgg 77 48147627 Coding 3 594 ctgcgtgagtattttatact 12 49 147628 Coding 3 609acaatcatcttccttctgcg 0 50 147629 Coding 3 611 tcacaatcatcttccttctg 0 51147630 Coding 3 623 cactgaatctcgtcacaatc 49 52 147631 Coding 3 628actggcactgaatctcgtca 85 53 147632 Coding 3 662 tactgagaattcagtgagga 0 54147633 Coding 3 665 cagtactgagaattcagtga 58 55 147634 Coding 3 670aaacacagtactgagaattc 2 56 147635 Coding 3 679 cttctgctgaaacacagtac 67 57147636 Coding 3 698 ccccacacatgtaagactcc 0 58 147637 Coding 3 702aacaccccacacatgtaaga 68 59 147638 Coding 3 757 cttttatactgctattgaaa 0 60147639 Coding 3 785 gcagcaacaactggaatcca 83 61 147640 Coding 3 793gtagtaaagcagcaacaact 5 62 147641 Coding 3 809 ctaagcactagaaagagtag 79 63147642 Coding 3 912 taaagtagcacttcttacca 42 64 147643 Coding 3 923ggttttgtctctaaagtagc 64 65 147644 Coding 3 964 atggctggtatgacgtgatg 7166 147645 Coding 3 1013 gttgctggagacaacggctc 76 67 147646 Coding 3 1017aactgttgctggagacaacg 85 68 147647 Coding 3 1057 gttccacttttcctggattg 069 147648 Coding 3 1070 agttcttctgtatgttccac 86 70 147649 Coding 3 1072aaagttcttctgtatgttcc 71 71 147650 Coding 3 1135 gatggctgcccgggaccacg 072 147651 Coding 3 1138 tcagatggctgcccgggacc 83 73 147652 Coding 3 1163gaagaactctctctctctat 0 74 147653 Coding 3 1175 ctacttaaaggtgaagaact 0 75147654 Coding 3 1183 actggttactacttaaaggt 5 76 147655 Coding 3 1220gagtgatacgagtttaaagc 55 77 147656 Coding 3 1247 gagtgatcactctcagaaca 2978 147657 Coding 3 1251 tctggagtgatcactctcag 78 79 147658 Coding 3 1293gctatgtgattccagacagc 0 80 147659 Coding 3 1316 ggaaattctgagtcagataa 0 81147660 Coding 3 1369 ttacggttatgagctcttgt 10 82 147661 Coding 3 1400ttatcataaccaaaggaggt 49 83 147662 Coding 3 1435 tatcatccacaagtagatcc 4184 147663 Coding 3 1439 ccgctatcatccacaagtag 0 85 147664 Coding 3 1446ctctttaccgctatcatcca 63 86 147665 Coding 3 1451 aaggactctttaccgctatc 8787 147666 Coding 3 1474 cttctgttggtctataacca 0 88

As shown in Table 1, SEQ ID NOs 11, 12, 13, 16, 17, 18, 22, 24, 25, 28,29, 30, 41, 44, 45, 48, 53, 55, 57, 59, 61, 63, 65, 66, 67, 68, 70, 71,73, 77, 79, 86 and 87 demonstrated at least 50% inhibition of humanInterferon gamma receptor 1 expression in this assay and are thereforepreferred. The target sites to which these preferred sequences arecomplementary are herein referred to as “active sites” and are thereforepreferred sites for targeting by compounds of the present invention.

Example 16 Western Blot Analysis of Interferon Gamma Receptor 1 ProteinLevels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to Interferon gammareceptor 1 is used, with a radiolabelled or fluorescently labeledsecondary antibody directed against the primary antibody species. Bandsare visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, SunnyvaleCalif.).

88 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcgctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2atgcattctg cccccaagga 20 3 2059 DNA Homo sapiens CDS (44)...(1513) 3ccgcaggcgc tcggggttgg agccagcgac cgtcggtagc agc atg gct ctc ctc 55 MetAla Leu Leu 1 ttt ctc cta ccc ctt gtc atg cag ggt gtg agc agg gct gagatg ggc 103 Phe Leu Leu Pro Leu Val Met Gln Gly Val Ser Arg Ala Glu MetGly 5 10 15 20 acc gcg gat ctg ggg ccg tcc tca gtg cct aca cca act aatgtt aca 151 Thr Ala Asp Leu Gly Pro Ser Ser Val Pro Thr Pro Thr Asn ValThr 25 30 35 att gaa tcc tat aac atg aac cct atc gta tat tgg gag tac cagatc 199 Ile Glu Ser Tyr Asn Met Asn Pro Ile Val Tyr Trp Glu Tyr Gln Ile40 45 50 atg cca cag gtc cct gtt ttt acc gta gag gta aag aac tat ggt gtt247 Met Pro Gln Val Pro Val Phe Thr Val Glu Val Lys Asn Tyr Gly Val 5560 65 aag aat tca gaa tgg att gat gcc tgc atc aat att tct cat cat tat295 Lys Asn Ser Glu Trp Ile Asp Ala Cys Ile Asn Ile Ser His His Tyr 7075 80 tgt aat att tct gat cat gtt ggt gat cca tca aat tct ctt tgg gtc343 Cys Asn Ile Ser Asp His Val Gly Asp Pro Ser Asn Ser Leu Trp Val 8590 95 100 aga gtt aaa gcc agg gtt gga caa aaa gaa tct gcc tat gca aagtca 391 Arg Val Lys Ala Arg Val Gly Gln Lys Glu Ser Ala Tyr Ala Lys Ser105 110 115 gaa gaa ttt gct gta tgc cga gat gga aaa att gga cca cct aaactg 439 Glu Glu Phe Ala Val Cys Arg Asp Gly Lys Ile Gly Pro Pro Lys Leu120 125 130 gat atc aga aag gag gag aag caa atc atg att gac ata ttt caccct 487 Asp Ile Arg Lys Glu Glu Lys Gln Ile Met Ile Asp Ile Phe His Pro135 140 145 tca gtt ttt gta aat gga gac gag cag gaa gtc gat tat gat cccgaa 535 Ser Val Phe Val Asn Gly Asp Glu Gln Glu Val Asp Tyr Asp Pro Glu150 155 160 act acc tgt tac att agg gtg tac aat gtg tat gtg aga atg aacgga 583 Thr Thr Cys Tyr Ile Arg Val Tyr Asn Val Tyr Val Arg Met Asn Gly165 170 175 180 agt gag atc cag tat aaa ata ctc acg cag aag gaa gat gattgt gac 631 Ser Glu Ile Gln Tyr Lys Ile Leu Thr Gln Lys Glu Asp Asp CysAsp 185 190 195 gag att cag tgc cag tta gcg att cca gta tcc tca ctg aattct cag 679 Glu Ile Gln Cys Gln Leu Ala Ile Pro Val Ser Ser Leu Asn SerGln 200 205 210 tac tgt gtt tca gca gaa gga gtc tta cat gtg tgg ggt gttaca act 727 Tyr Cys Val Ser Ala Glu Gly Val Leu His Val Trp Gly Val ThrThr 215 220 225 gaa aag tca aaa gaa gtt tgt att acc att ttc aat agc agtata aaa 775 Glu Lys Ser Lys Glu Val Cys Ile Thr Ile Phe Asn Ser Ser IleLys 230 235 240 ggt tct ctt tgg att cca gtt gtt gct gct tta cta ctc tttcta gtg 823 Gly Ser Leu Trp Ile Pro Val Val Ala Ala Leu Leu Leu Phe LeuVal 245 250 255 260 ctt agc ctg gta ttc atc tgt ttt tat att aag aaa attaat cca ttg 871 Leu Ser Leu Val Phe Ile Cys Phe Tyr Ile Lys Lys Ile AsnPro Leu 265 270 275 aag gaa aaa agc ata ata tta ccc aag tcc ttg atc tctgtg gta aga 919 Lys Glu Lys Ser Ile Ile Leu Pro Lys Ser Leu Ile Ser ValVal Arg 280 285 290 agt gct act tta gag aca aaa cct gaa tca aaa tat gtatca ctc atc 967 Ser Ala Thr Leu Glu Thr Lys Pro Glu Ser Lys Tyr Val SerLeu Ile 295 300 305 acg tca tac cag cca ttt tcc tta gaa aag gag gtg gtctgt gaa gag 1015 Thr Ser Tyr Gln Pro Phe Ser Leu Glu Lys Glu Val Val CysGlu Glu 310 315 320 ccg ttg tct cca gca aca gtt cca ggc atg cat acc gaagac aat cca 1063 Pro Leu Ser Pro Ala Thr Val Pro Gly Met His Thr Glu AspAsn Pro 325 330 335 340 gga aaa gtg gaa cat aca gaa gaa ctt tct agt ataaca gaa gtg gtg 1111 Gly Lys Val Glu His Thr Glu Glu Leu Ser Ser Ile ThrGlu Val Val 345 350 355 act act gaa gaa aat att cct gac gtg gtc ccg ggcagc cat ctg act 1159 Thr Thr Glu Glu Asn Ile Pro Asp Val Val Pro Gly SerHis Leu Thr 360 365 370 cca ata gag aga gag agt tct tca cct tta agt agtaac cag tct gaa 1207 Pro Ile Glu Arg Glu Ser Ser Ser Pro Leu Ser Ser AsnGln Ser Glu 375 380 385 cct ggc agc atc gct tta aac tcg tat cac tcc agaaat tgt tct gag 1255 Pro Gly Ser Ile Ala Leu Asn Ser Tyr His Ser Arg AsnCys Ser Glu 390 395 400 agt gat cac tcc aga aat ggt ttt gat act gat tccagc tgt ctg gaa 1303 Ser Asp His Ser Arg Asn Gly Phe Asp Thr Asp Ser SerCys Leu Glu 405 410 415 420 tca cat agc tcc tta tct gac tca gaa ttt ccccca aat aat aaa ggt 1351 Ser His Ser Ser Leu Ser Asp Ser Glu Phe Pro ProAsn Asn Lys Gly 425 430 435 gaa ata aaa aca gaa gga caa gag ctc ata accgta ata aaa gcc ccc 1399 Glu Ile Lys Thr Glu Gly Gln Glu Leu Ile Thr ValIle Lys Ala Pro 440 445 450 acc tcc ttt ggt tat gat aaa cca cat gtg ctagtg gat cta ctt gtg 1447 Thr Ser Phe Gly Tyr Asp Lys Pro His Val Leu ValAsp Leu Leu Val 455 460 465 gat gat agc ggt aaa gag tcc ttg att ggt tataga cca aca gaa gat 1495 Asp Asp Ser Gly Lys Glu Ser Leu Ile Gly Tyr ArgPro Thr Glu Asp 470 475 480 tcc aaa gaa ttt tca tga gatcagctaagttgcaccaa ctttgaagtc 1543 Ser Lys Glu Phe Ser * 485 tgattttcctggacagtttt ctgctttaat ttcatgaaaa gattatgatc tcagaaattg 1603 tatcttagttggtatcaacc aaatggagtg acttagtgta catgaaagcg taaagaggat 1663 gtgtggcattttcacttttg gcttgtaaag tacagacttt tttttttttt taaacaaaaa 1723 aagcattgtaacttatgaac ctttacatcc agataggtta ccagtaacgg aacatatcca 1783 gtactcctggttcctaggtg agcaggtgat gccccaggga cctttgtagc cacttcactt 1843 tttttcttttctctgccttg gtatagcata tgtgttttgt aagtttatgc atacagtaat 1903 tttaagtaatttcagaagaa attctcgaag cttttcaaaa ttggacttaa aatctaattc 1963 aaactaatagaattaatgga atatgtaaat agaaacgtgt atatttttta tgaaacatta 2023 cagttagagatttttaaata aagaatttta aaactc 2059 4 25 DNA Artificial Sequence PCRPrimer 4 cttagaaaag gaggtggtct gtgaa 25 5 21 DNA Artificial Sequence PCRPrimer 5 cctggattgt cttcggtatg c 21 6 26 DNA Artificial Sequence PCRProbe 6 cgttgtctcc agcaacagtt ccaggc 26 7 19 DNA Artificial Sequence PCRPrimer 7 gaaggtgaag gtcggagtc 19 8 20 DNA Artificial Sequence PCR Primer8 gaagatggtg atgggatttc 20 9 20 DNA Artificial Sequence PCR Probe 9caagcttccc gttctcagcc 20 10 26000 DNA Homo sapiens 10 ctctaaattcttatcagtga cccaattata gcttagccag ccataaattc aactatacct 60 gcttcagtcccacacctctt tacaagcttg ttttgttcat catatattta tttaataagt 120 ggccacagacattggttcag ctgctcaatg ctgacagtga tgattcagga ttacatacaa 180 ttgatgattcaggatcatca atattccata aatattaatg gagtaatagg cacttggcta 240 ggtgctcggggtctgacagt gaagaaaaca aatttatttt ttagaggaag agggagacta 300 ctgaaacaaaattttctacg tgtgtatata catattttat gtatgcacac acacacggat 360 gcatttcattaaatctcaga ttaagtccct gtttacgcac ctataatcgt gttgctctcc 420 ttggtaacaattactacaat ttcagtaatt atttgtgaaa tatatatata tataaaatgc 480 atatatttaacacatacaat ttattcatat ttaattcaaa tttaatagat atattaaatt 540 tccagtagaatctccagata gtagtaagta ctatggaata aacaaagtta aataagggcc 600 agtgtaaaggcatgggtaga gtgtgagagg gcaagagtat tggctataat ttacagacta 660 atcagcggaatttaaactat atgcttcagt tacaaatctt cacgctgtct cctttaaagg 720 gaagctggcagacatttctg ctttggagaa aagaggaagg aaatgcactg tgctcagagc 780 caaaatgcacagtttgttcc acacacacag gctctgttct cattggatca tatttctagc 840 ttcttaaattgttttatttg gcctattaac ttgaaaggca ggtgagtatg gtcttcaaaa 900 tgtagccttgctcctgagca gcagcacttc agtatacact tggctttgga atgatctacg 960 ctccaccccaatttgttttc ttcattttaa aaactaatct gaggctctag tggggaccaa 1020 cgtatacttggatattcccc agccattagg cattggaaat ttatcaagga aagctggcac 1080 cgctcatttgtttctgtgtt agctgtatga gatttttata tgggatttta aaatacagtt 1140 tattgctcttcttcctacct ctcttattag ccggtggcca cccccatctt ttaactttat 1200 tcaggctaattttaccttat ccttcaggtc acaacataat ggtaacttcc ttaaaaaaga 1260 atttccctaaatccagacac agtggctcac acctgtaatc ccagcatttt gggagcctga 1320 ggcaggagaatcatttgagc ccaggagttt gagaccagcc tgagcaacac agcaagaccc 1380 cagctctataaaaaatttaa aaactagccg ggcataaggc caggcgaggt ggctcactcc 1440 tgtaatcccagcactttggg aggccaaggt gggcggattg cttgagctca ggagtttgag 1500 atcagcctgggcaacatggt gaaaccccat ctccacaaaa gacacaaaaa aatgcctgtg 1560 gtcccagctacttgggaggc tgaggtggaa ggatggcttg cgcccgggag gtggaggttg 1620 cagtcagctgagattgtgcc actgtacctc agctaggtga tacagtcaga ccctgtctca 1680 aaaaaaaaaaaaaaaattag ccaggcatgg tggtgtacac ctgtagtccc agctatttgg 1740 gaggctggggcaagaggatc atttgggccc aggagtttga ggctgcagta agctatgatc 1800 atgcctctgcactgcagcct agacgacaga gtgagaccct gtctcaaaag aaagaaaaaa 1860 gaaaagaaagactttcttta aaatgtagac taagttaaat tcctgtttat actcctttaa 1920 tctcttcccattgctctcct tgggaatgat gatcacgatt tcagtacttg tgtaattcct 1980 tgtttaacagctatctccct cagtagacag taagctccgt gagggcaggg gccatgtctg 2040 tcttgctcaggtctttatct ctgtgcctgg tcatgtcagt cacagacagt atcacttgat 2100 aatattcacccaattaataa atatatttag tctccaaatg actaactagc aaagtcttca 2160 gaaggatacgtagggtggtg ggtgctgtga ttgtgaaact tgcccagatc cctctcagga 2220 acagaagactgggttctgtc cccttcctga tgctaggagg gctgccagta gacaccctca 2280 accgtcagcgttcctctggg gaatgcctcc atgaaagaaa tggacagccc caaaatcatg 2340 acagttggtattcaatgact aatcagtgct gggctttaag acccagcccc tcataccaac 2400 tcaagacacctttacaaggc catcccaact ccagagcgcc ccttgggagc ctttactggg 2460 atggcatcacagatcaactt cttcctccgc tctgcttcct ccacctcccc ttcacagacg 2520 tttattccaagagcactccc taatcaatca atgccttata ccttaatctc gacctcagag 2580 tttactgccagggtaaccca gcctgtgaca agtaccacaa aaatgtttac aaactggata 2640 tcatattttgtcctactcca tagacgccta aacggtaaag atcagttatt tatccttgca 2700 tccttacaacgactagaagg tgtctttcat atagtggaca tcagaaacac atcaaaggaa 2760 atattctccctaaacttagt ctagattgag ctaaattctt ctgattcaaa gtgtggtcct 2820 gttgaaaggggcaccagcat cccctgtgca ttttgtccga aatacaaaat ctcaagaccc 2880 aacctgaattagaactactg aatccaaatc tgcattttat caagatcccc agatggttca 2940 catgtatgttgaagtttgag aagtactgag ctaagagatt tattaataca agtaaaatga 3000 tctggctgataatacctcta gtttatatag tgtctttata aggattttcc ctctttaaaa 3060 attttccttaacactagtaa tcatgcagtc tttcactcaa caaatactaa ttgagtgcca 3120 agtaaagatgtcagctctac ggagcttaca ttttagaagg agaagaaaac aaatatcaag 3180 gtgtatattatagtgtacag caaggtgaaa agtgccatga aaaaacagta gggcggggta 3240 aggaggaggagaaggtgtgt tgcaatttaa aatttttaac aaaagttcat gatactcaga 3300 gaattgaaacaggtgagatc attagacatt cgcatgtttg agcacaagcg ctgaaggact 3360 tagcaatgtggctgcagtgg aataggagag atgcaaaata acagacaaac ccagagaggt 3420 aagagagcagacctcttcat gagaggctgc ctgataaact gatttgacac tgaattgctg 3480 agaaggtaatgcaaatagtt tttttctcca agaacaaagt agttcttggt caagccgatt 3540 tgatttgggctgtgggaatc tgcacaaacc atttcactag ctaagtctca ggttcctcaa 3600 atgaaaaagcagggactgga aatggctttt gaagtctcat tctcttctaa aaatgtcagg 3660 ctccaagacaaccaggtgaa gtccaagagt tagtaaaata aggattgtgg ctcggctgtg 3720 gcctaatgcaaacttgcaca accccaggaa accgaaaaaa actggaagaa gaattgcaga 3780 atggggtgccaggttgaaag accttaacct ttgcactcaa attcctccca cacccagaag 3840 tccaggtcccgaccgcacga cgccgtgctc actgctgggt gctgcgcctg agtccgcctc 3900 ctgcggcttcccggacttga ccccgcccac gccctggtcc cgcctcctgc cgacgccggc 3960 acagaccccggtgacggaag tgacgtaagg ccggggctgg agggcagtgc tgggctggtc 4020 ccgcaggcgctcggggttgg agccagcgac cgtcggtagc agcatggctc tcctctttct 4080 cctaccccttgtcatgcagg gtgtgagcag ggctgagatg ggcaccgcgg atctggggcc 4140 gtcctcaggtaccgtcgttc gcggcagggc tgcggccggg tcgggacgag agggagggag 4200 ggatccgccccagccgggaa gccccgcccc gcttctccga ggtcgcccta gcccgggacc 4260 cctgcgtcgggccctgagcg ggcgacgggg acgcgaggtg gggtcgcgag actggggctc 4320 cgtggtttgaactcgtcggt tgcctttctc ctgcctcttc cctggttgcc gccacaacaa 4380 atcacgccgcttgtttttcc gactcttgta atccactttt aaatacgatt atgcgctgca 4440 gaaggacgttgctggtcaat gtgggacagc gtggtctcaa agattataat atcgtcttct 4500 tactgtacctttttttatgg ttagatacgc agatactcac tagtatgcta cggttgcctg 4560 cagtgttcagtgcagtgaca ggctgtacag gttcatgacc taagagcagt aggctgtgtc 4620 atctgggtttgtctgggttt ctactgcccg aagaaattgc ctaaggggac atttctccat 4680 ttctcagaacggatctccat cgttaagtga cgcatgactg tgagtgtcct gatcagcatg 4740 tggcatcgtggtctcagttc gtggcttaaa tacttctttc ttagcggggg tggggggggg 4800 gactcaaagtgcttagggta ggaaagtatg cattatgtcg ccagctgaga agtaagtggc 4860 agctgagaagtaagtggcag tccgtcttag gtctcagtct ggaagtggtg gattgccggc 4920 actcactagcagatttggca gtaggtctct tcgtacgggt ttgttcttcg gagcatttta 4980 tttccacctgcttattgaca tttcctagaa aacaaatgtt gagggtggct tttctgaagt 5040 gcagagtaattgtgattacg agctccagag aaaacagtaa accttttcaa ttcagttttc 5100 ttttttttattatgatttat gattcatagg gactttgaaa ggtccacgtt ttggttctgt 5160 gttcacaacaatcttctgga gtaggcgtta tttgtctctc tctttatgga cgagaaattg 5220 agttacggagaggcctgccc aactggcaga gaggtcatag ccactcagtg gggagctgct 5280 caatttggctccagagtcca agttcttaac tgctcttcta ggctctagtc agtcatccac 5340 atgggcagttgcagtacttt gcggtaagag cttgcactta ggcacagact agtggaaagc 5400 aaggcggggtgcttgtctct tttagggaag tcagtgaaga cttcacaaag gtgaccccag 5460 tgttggaaccagaggtgcag agactgaggt gcagaggtgt gatagcatgt tgaattcgtg 5520 gaatattaagcgttctattg taaagtgtgt gtgagagctt acaaggggag gagataggtt 5580 aggtggagaagtaggaacca aataatgaat tgccatatta tgtgtaccag caagaaacct 5640 agctttattaacttaagcaa aaagaggaat ttattgtctc agaaaaacca gagttaaaca 5700 gaaaattatagatgccagct gggtctcaag aggctagaac caggaaccca aactgctagg 5760 actctccatttcttatttct gtctgggtat tgacagcttt ccacacttgg caggaatcat 5820 ggatgctgacagcttctgag ttttgacatc ttgcagcctc ctccattcta acaaaatgcc 5880 agggaaaggctctgatttgc ccagctaggt tcaggtgcca gcttgcatgg gaaggacatt 5940 ctcgtagagggttgggttgg gttaccagaa gaagcggaat ggcgtgcctg aagtacaggt 6000 gttgagccaacaaaataata cacccactat gccttgctga agtcagtcat tatttactaa 6060 tgctgacatcattggattat caattacaaa gggaaatgat tttatgcagg atcagacatg 6120 ccatttagaaagaccagtca tcttataaaa tagtttgagg gtaaacacaa ggcccattag 6180 gaggctgctgcttaagttgg agtcgataag tagtttaagt ggagaagagg acagttagca 6240 gaacatttagaaggaagagt tagtaggcct ctgactggat gtcagcagag aaagaggata 6300 acgctagcgcaggcatagcg ttaaagcaag tgccaggccc ttccttctga atacggggcc 6360 ctatgagtgcaccgtgtgac ccctgtgagt gcacaatttg cacgtccttg tagcttgctc 6420 tgggaggaggattgcctgtc tgcctcccgt gggaagatca actgcagcct tctatattct 6480 gcagaaacttttgaatttgt ctctgtacct ctcgttctaa aactggggtc acttttgtga 6540 agcctttcctgactccccta gaagagacaa gcaccccggc cttgcttttc attagtttca 6600 gtgagccctctttttaaagt gcatttctaa cacatttctc tagatttttc agtgcatttc 6660 tctagattttgttgtgttcc ctgttctttt aaagttaatt taaaacacaa aatctccaaa 6720 gtaggaagaaaccaacaatg ctctggctct tgcttagctt cccagcctta ttcttactgt 6780 tgctgggcttgactctatag ttgaagttca tcttagatga gaggacctgg taagaacagg 6840 gaagcagacaagtgaatatg gtgatatagt cagtctccag gaaatccagg tgtttaggaa 6900 tgtattgtgttacattcatg ggctttcatt aatacttctt aagtgaaaat aaaatctgtg 6960 tgtgccacttaatcagtagt atttcactct tcatgcactt ttacacataa aagtagtata 7020 tatttttaagtgaatcaggg tgtgccactg gctttcttca tccagatgag tgtcccactc 7080 tgagtaatagctgaagagct aggctggact tcacaccaac tggaaggctc ttcactgacc 7140 ttgtacagtttatcagctat gcttcctact aattatctga ctttaggata aagctctgag 7200 tactgtttaaatttttcact atttctttgg attctatagt tatttcagag attcaaaata 7260 cacaggataatactttttaa ttatatttat tgaatagggt attaaatttt ctttttcatt 7320 ttattctgattcataattca cacagacttt gaaaggacca tattttggtt ctatctaaaa 7380 gaaaacctaggctgggggca gtggctcatg cctgtaatcc cagcacatcg gcggaaggat 7440 cactggagcccagaagttca agaccagcct gggcaacatg gcaaaaccca tctctacaaa 7500 aaaatacaaaaattaaccct gtgtggtggc atgtgcctgt agtcctagct actggggagg 7560 ctgaggtaggaggatcactt gagcccagga ggtctaggct gcagtgagcc atgattgtac 7620 cactgcactccagcttgggc aacagagcaa gactctgtct ctcgtgtttg tgtgtgtgtt 7680 tgtgtgtgtgtgtgtgtttg tgtatgaaaa cctaaagaaa agtaataaac agctatctta 7740 caaaatttggcagcatacca gtagtgaagt tgctttacaa accagatgag taattaatgg 7800 ttcaattttgattgaacaaa gaatcagaat gaggtttgaa ccagaatgaa gtgaattcct 7860 aacagaaaggaatttacttg taattttctt attaaagggg tgcctttatt ttagattatc 7920 ctaatgcatagtaatcagaa tactaggtat ctgtttaggt cttgtgtatt tttaagaaat 7980 tattcattttaatgcattgt tcaacttttg cagtggccat aatataattg tgataatgta 8040 ataaaatcaggaaacattac ctgaagcaga tgcttttgaa gtcaccagaa aatagtcttc 8100 tccaattccaatttaaatat ggaggtaatt taccccttat tttattaaac gatcacaaag 8160 atgttggaagcatttttctt aatgaaaacc aacaaaactt tgcatattgt caagttacgt 8220 aacttctctaagcctttgtt ttctcattgg taaaatatgt atggtaacat tgtctccctc 8280 acgggatggatgtgatgagt atctgaggta atgcattaaa aaggtaagca cactgtgtgt 8340 cttgtagtaaatacttaata tgttagggat tattcaataa tgtgcattat tattttaaca 8400 aatatatctttaaatattaa attcccttag tcatatgtga gaaattgtgt tggcttttaa 8460 aaaaacaaacttaaccagat cagtatgaga aacttaaaac aggctcagca gctaactcat 8520 tcttccatctcttccaacag acaaaaaagt ttggtttttt agtagactgg aaaggcctgc 8580 attgttttttcatttcctca actgtttttc aattcagaat gagttagtcc aaaagagaat 8640 tagtgttgagaaatgacttc tctcagggac tagattggtc tggtccagtc agaactaatg 8700 ccagaccaaacccaactaga tgaaattgga taaataggag taagaataaa gccctggatt 8760 tcaagtcagaaaagtggctg agcagatatt tattggataa gccccaactt ggtggtggtg 8820 cgagggaaaaatatagggga ggttagttac ttacaagttc actgcaggcc actatgtgtt 8880 atgcctggctctttaaaaag aaaaatttca attagaagat ttcaatgaac acttgaaaca 8940 tcacctgcaagaattatcaa aatgtctgta ctcagcttaa acctggggga tgcggctcct 9000 tgctacggtgaaagttggaa agcattaaga aaaatagtca ttttatcacc actggaagcc 9060 attcgggagggagattgtaa tcaaatatgt tctgacaaaa atacaaaggc cttgatttgg 9120 tacttttcatttattcagtg tggatttctt atgctggttc tgtttgctgt gcagatgaaa 9180 cctttgtgacagtccccatt cctgtccctg tatctttaac atggggaagg tgttagatct 9240 tctctgagagttgttcttaa ggaaaagatc cagatgcttt aatttgaaac atttgttttt 9300 gagttgtagtgtcagatgac ttagaatcca gaacaacagt ttacttcatc ctcacttcct 9360 tatgcttctcagcgtgctct tcccatgaag aggttggtga gcaggttgtc agatgatttc 9420 tgcagagtccatagccctgt tccaccatag atgtacaaac cttttataca gtctttttac 9480 actagtgtgttaagatgggt gatgaactcc tcgggctgtc cttttttaaa gagacacaga 9540 tgcttttggtggttacagga tggactcagg tagcagccct ggttcctgtc acccagctat 9600 gcagaggaaatggccagtgt cttcagacta tactctgcag gtatctgtag tttgccatcc 9660 accctacattcaccaacatt tattgcagag ctatcacgac tgctattttt tggtcctttg 9720 tgacattgttatggtcccgt cgatagcact gctctgtatc acctgctgat cttcactggt 9780 cttttgtgttgtctctcttt tttcatatcc tgtctcttct ggcttatcag ctttctgagg 9840 aaacagttggctttaaccag ggccctcctt tttttttctg cattgtaatt aaagtgttag 9900 gaaaaacaaattcttctctc ctgtgtttag ttttcttaaa ttatcagctg tgagttgtag 9960 gggagttctgggaattagat cttaaaatag ctaaagttta ctccggatag tatgcagatg 10020 taatacactaaataattctg agtccatttc caaaagcaac atgagttaca caaaactatc 10080 ttggcattttcaagagatgg gaaaatctgt aagattatct tatttcatga aatcactctg 10140 gtaagatcttttcttaagcc ccagagataa taatactctt ttgctgtttc tacatatatt 10200 tttatttacatagtgtaaat gtattagaag ttaattgtaa gccttaatta atttaatctg 10260 aatatataacaatttaaacc tcacattaca aaattctgaa gcttaagaaa actgcttatg 10320 tctgtcatattattttggca aataatattt ggcaaaaaat aatatttggc aaaacaaagg 10380 cagatgtctaataaccacac agtttctcct ctggtagcat taaaaaccac agattacatc 10440 ttttggtgtaaatttataag tttttctttt aacctagtag taatttcttt ttgctataaa 10500 ccattgaaatattttcctcc taccacctgt ctgtcttgct ttgaaagcta tgatttaagt 10560 ccttgtcctagtctaaccat atcaggcttt tacttaaatt ataggaaaca tactttgatt 10620 cacgttaaaaactaatctaa tgctgcacaa gcctactcct gtttgtagtt ttccattgtg 10680 tgattcagtagccccttcct aagtacaaat gcactttgtt tgatattcta gtatgcagat 10740 tgacttctatatgccttttt ctcacaggaa tttcaggttg agcgtatacc attataaagc 10800 cattcctactgggtataatt aaaaagagag ccttaggctg ggcccggtgg ctcacacctg 10860 taatctcagcactttgggag gcccaggtgg gcgattcatg aggttgggag tttgagacca 10920 gcctgaccaacatggtgaaa ccccatctct actaaaaata caaaaattag ccatgtctgg 10980 tggtgcaaacctgtaatctc agctactcgg gaggctgagg caggagaatc gtttgaaccc 11040 aggaggcagaggttgcagtg agcagagatc atgccattgc actccaacct gggcaacaga 11100 gcaagactctgtctcaaaag gaggggagtg gagcttaaaa ggaagcaaag gatttccagt 11160 cttggaaacctagaggaagg ggggtccttg ctatgtagtg gcagaaagct gagtgacact 11220 gtagccttcagtgacaccaa aagtagagaa tgtctgagga gctgtgtgat ctagctcagg 11280 aggtttctaaccagagtgct gaaagtgcca cctggtttct tcttgcgtct tatagtaaca 11340 ggtgagactttgctcaatca tgggaagaac tgttaaaaag aaaccaggac cacctggttt 11400 gaaaattctcagcctctcga gaggcaaaaa aatgccaaaa tgaagaaatg gtctccgaag 11460 gaaggctcagatttagtatg agttttctta gcatcaagta cctctgtttt ccggttgaaa 11520 aagtatatttatatgtgtga ttgttatttg atattaatgt tgattgaacc caggtgaaga 11580 ggaatgacttttatttctac ctaaaatatg gattgtgaag tatttgagtt gtttcaggag 11640 gttgagatgcaaacgtgtga tagaaaaaag accctcttaa ggaagcctca gtatcccctg 11700 ctatgaatggccatgggaac tctacaaaaa acaaagagcc aaggctactc tggcaagaac 11760 agtgccttagggatcaccta caaggaagtt gcttagagaa ccagagatgg caggtaccaa 11820 gagccttcagctgcgcctct ggaagcagaa ggagaaaacc taggtgtctg gtcactggcc 11880 acaaaaaggtcaagttagta gtatgaagta tttgaatctt cagaccctcc ccagcttgat 11940 aatctcaaccttttggccca aattattaca ccattttggt aaataatata gaagttagat 12000 cttttggcaataaccaaatc tattcatttg tttgagggga gccatgaaat atacaggttt 12060 actgtaaagcattctgtttt taagttagtt tttcctcaac ttttttttta aagaacactt 12120 aaattgtaaacataaagcag ggtttctcaa ccttggcact gctggcattt ggggctatat 12180 tattttttgttgtggaggct gtctgatgca ttgtaggata tttagcagca cccttggtct 12240 ctgccctctagatgccagca gtacctcccc cacacacttc ttctccaggt tgtgagagcc 12300 aaaaatgtctacagacattg attgcctaat gtctcctggc aacaaaatca cttcccacag 12360 agaaccactgcaataaagga aaaaataatc agaaaaggaa aataataatg gtgatagcat 12420 ttaattgtccattagtgcct attatttacc taaaaaactc ttaatccatg ttctgctttg 12480 aaaatatatgtgttaaataa ttgtaaatta aggcaatata ccagtttggt cttgaatatt 12540 ggaatttttgtttggggaaa atatagattt tcccttcagc agtaagcaaa tatcttaggg 12600 atatgaaagtaacacctcat gacagatcag gaaatctgaa gtatttagca gtgtggccaa 12660 gaaagaacccaaatttatta atgtttgacc tagggagtat ttttcttatt taagcagtct 12720 gcttgatataaatataaatc atcatcatta tcatccaagg cactttggaa aaatagaaac 12780 ttctgtccctctcacaattt ttgaggatga ttataagtta tgtttggaag ccactgttcc 12840 tatccacatggacattcatt tatttgtttt agttatataa gtaaatcatg tttaatagag 12900 tgttccagggaggtgagatt gaataaccat aaatttgggg tcacatcgct aactgtgtac 12960 attcatgtttatctgtaaag aagtgaaggg agaaaggaga agtgtgtgtg ttcagttttc 13020 caaggggtgttggataacaa ccttttacac caaacagtgg ggaaaaatga ctcctttttt 13080 tttttttctaagagaactga atgcagtggc acagtcacag ctcactgcag ccttgacctc 13140 ctgggctcaagcaatcttcc tcagcctccc gagtagctga aactacaggc acaggcctct 13200 tcacctggctaattttttaa ttatttgcgg agacagggtc tccaactcct gagctcaaca 13260 gatcctcccacctcagcctc ccaaaatgct gggattacag gtgtgggcca ccgcacctgg 13320 cccatgactacttttaataa ctagattttg gtagtgtctg attagatgtt aaaatgactt 13380 ctgtctgttctcttacgtga ctcaagtctt atcaacagat gttttctgaa agagtgaagg 13440 caagaagaaatgttgggtat gttttgtaaa aagccctgca caccccagag agcagagtgt 13500 tgatgaaactggaatgtgtg gctccattgt tcaatcttta ggtatctatt cctgtactat 13560 agaaaaagtgtggaaggcat aaatagtagt atgtgtagag ttgagaggtt gatggtattt 13620 tagagtgaaattggccatcc tcattgtagc cagttcctct gtagacattt gtattagtct 13680 gttttcgtgctgctgataaa gacataccca aggctgggaa gaaaaagatg tttgattgga 13740 cttacagttccacatggctg gggaggcctc agaatcacag caagggcgaa aggcacttct 13800 tacatggtggcggcaagaga aaaatgagga agaagcaaaa gcagaaaccc ctgataaacc 13860 catcagatctcgtgagactt actcactatc acgagactag catgggaaag accagccccc 13920 gtgattcaattacttccccc aggtccctct cataacacgt gggaattcca agagatacaa 13980 ttcaagttgagatttcggtg gggacacaca accaaaccat attatgctac ccctggcccc 14040 tccaaatctcatgtccttac atttcaaaac caatcatgcc ttccaaacag tctcccaaag 14100 tcttaactcatttcagcatt aacccaaaag tccacattcc aaagtcccat ctgagacaag 14160 gcaagtcctttctgcctgtg agcctgtaaa atcaaaagca agctagttac ttcctagata 14220 cagtgtgggtacaggtattg ggtaaataca gccattccaa atgggagaaa ttggccaaaa 14280 caaaggagttatagagccca tgcaagtcca taatccagtg gggcagtcaa atcttaaagc 14340 tccgaaatgatctcctttga ctccaggtct cacatccagg tcatgcggat gtaagaggtg 14400 gcttcccatggtcttgagca gctctgcccc tgtggctttg cagggtacag gctccctctc 14460 agctgctttcacaggctgcc atcaaatgtc tgcagctttt ccagaagttg tcagcagatc 14520 taccattctggagtctggag gatggtggcc ctcttctcac agctctacca ggcagtgccc 14580 cagtagggacttgtgtgagg gctccgacct cacattttcc ttccacactg gcctagcaga 14640 ggttctccatgagggccctg cccctgcagc aaacttttgc ctgggcatcc aggcatttcc 14700 atacatcttctgaaatctaa gcagaggttc ccaaatctca attcttgact tctgcacacc 14760 cataggctcaacaccatgtg gaagctgcca aggcttgtga cttccaccct ccgaagccac 14820 agcctgagctatatgttggc ctctttcagt cacagctgga gcagctggga cgcaaggcac 14880 caagtccctaggctgcacac agcacgggga ccctgggcct ggccacaaaa ccactttttc 14940 ctcctaggcctccgggcctg tgatgggaag cgctggcgtg aatgtctctg acatggcctg 15000 gagacattttctcgatggcc ttggggatta acattaggct tcttgctact tatgcacatt 15060 tctgcagccagcttgaattt ctccccagaa aatgggtttt tcttttctgt ctcatagtca 15120 ggctgcaaattttccaaact tttatgctct gcttccctta taaaactgaa tgcctttaac 15180 agcacgcaagtcacttcttg aatgctatgc cacttagtaa tttcttccac cagataccct 15240 aaatcatctctctcaagttc agagttccac aaatctctgg ggcaggggca aaatgccgcc 15300 agtctctttgctaaaacata gcaagagtca cctttgctcc agttcccagc aagttcctca 15360 cctccactgagaccacctca gcctggattt tattgtccat attgctatca gcattttggg 15420 caaagccattcaacaagtct cttaagtaaa gttccaaaca ttttcctatt tcttctgagc 15480 cctccaaactgttccagtct ctgcctgtta ccctgttcca aagttgcttc cacattttct 15540 tgtatcttttcagcaatgcc ccactctgct ggtaccaatt tactgtatta gtcctttttc 15600 atgctgctgataaagacatg cccaagactg ggaagaaaaa gatgtttaat tgaacttaca 15660 gttccacatggttggagagg cctcagaatc atggcggggg agaaaggcac ttcttacatg 15720 gtggcagcaagagaaaatga ggacgaagca aaaagcagaa gccccaataa acccatcata 15780 tctcatgagacttattcact atcacgaaaa tagcacagga aagactggcc cccatgattc 15840 aattacctctccctgggtcc aagccacaac acatgggaat tctgggagat acagttcaag 15900 ttgagatttgggtggggaca cagccaaacc ctatcaacat tgaatacaat ctgtagtatc 15960 aaatattgaaaattctagtg atgaaatgag gattattaat tcaataatca gtgcatttga 16020 ttataaaacttctatcatat taagggaatt tattgcttca tgctgtattt gtgtttcctt 16080 tatattgcaatgatgtttcc ataatttaaa acgaatcaga actcttctat agatttttca 16140 ataaaattaatttctagatg tctactcttc atttaaataa ttttggtatc ctggtgaatt 16200 ctacttttcttcaaatatac atatctgggc aatgtggcat cttacaataa ggctttccaa 16260 tattaatgttagtttcttac aattgtccct tttacttttt tattttctta cagtgcctac 16320 accaactaatgttacaattg aatcctataa catgaaccct atcgtatatt gggagtacca 16380 gatcatgccacaggtccctg tttttaccgt agaggtaaag aactatgggt gagtgtcact 16440 cttttatttatcctttttat tccatttttg tttaggtcct tggaaattcc acaactgtgt 16500 tctttcatcagccttcccac gtggcaaaac attctaaact gcttatagag gtccaaaagt 16560 actaaagatgcaaattcttt gccaaatatt tcttgcctct tatttcctct tccttatcag 16620 tattgaaatagagaagtatc actaaacttc tgagattagc atgacaaata aagctaatag 16680 ttactatgtcatcttcctgt agtgtatttt tagtagaaca atataaattt caaaaaactt 16740 ttttgacaactgttagtttt tttaatgtgc ttacttctat aattaacata tataaatgga 16800 atttagagttgtttaaaaaa acagattttt aaaaaattga atgtggaatg tggcttgcat 16860 aggtcttacattttgaattg agcctcttta ctgtaacaga gtttgtagtt cttctaacta 16920 aagagtaagggtttcctact ttctctggcg tctccatcct attcttagct ctgctctttc 16980 taccgctttgtgctgtgaat aaaaagcaaa gcacagacag aaatggtttg agtttattta 17040 ataacaataaaagttatctt cgcatttttt tattcttttt agtgttaaga attcagaatg 17100 gattgatgcctgcatcaata tttctcatca ttattgtaat atttctgatc atgttggtga 17160 tccatcaaattctctttggg tcagagttaa agccagggtt ggacaaaaag aatctgccta 17220 tgcaaagtcagaagaatttg ctgtatgccg agatggtgag tagaatgtgt acacatgtaa 17280 attttaaattggaaaatatt tatacatgtc ttctgtatgt ttgcttttat tagcagttgc 17340 tgaaaattatcacaatgctt aagaaacact tggaggaatt tagagtcagt acaggttgag 17400 catcccaagtctagaaaccc agaatgctcc aaaatctgaa actttttgag cgccagcatg 17460 gcactcaaaggaaatactca ttggagcatt ttggatgttc agatttagaa tgttcaactg 17520 gtaaatataatgtaaatatt ccagaatcca gaaaaaaaaa ctcagaaacc gaaaatacta 17580 ctgatctcaagcgttttgga tatgggatac tcaacctgta tcctcccctt ctttcaagcc 17640 ccacaaaatagggctaacag gtggccatca cttagctgct tctatgccat gacatttcca 17700 cttcatacaattatgggtct gtgctgccca gcggtcccct gcaccaccct tgcccttccc 17760 atcttttgtcattcatttgt aagttatatt cactaaggac tttggcagct aactcttatt 17820 ctgaatgcctggggaacctg aataccacct taatacatta aaaaattgca gagttcctta 17880 ttctcttcagccactgtgat tttcaggtct tcagtcttcc agcatcccac tcccatagtg 17940 tacctgatgtttgctgtcac ctcaaactct tgaatcttta attccagttc ctgtattcca 18000 actatgacatcctgccttag ctctcagacc ctgactcctg ggtaaaagac acctttagtc 18060 acttgactcctccctagtct tctagccctt gggtcccttt ccctctagcc tcacttcctt 18120 tctctgctcctgccttttag attgtgccac ctcggtctct tctacaggct cttcctctcc 18180 ttaccctttaaatacttttc ttccccagag tccacaattt tgtcatatga ttaccacacc 18240 atctgcatgttagatttttt tccatataga tttatcaagg gcctactacg tgccggaaag 18300 tatactgggccctgaggata cactgatggg ttaaaaacag acctgcctgg cacacagtgg 18360 tacacagtaatatgtgaaga ctagataaat ggttcacaac aaagtctcaa acatcaaaga 18420 cctaataaatatttatggaa taagttctga tttttcacag taaaaagagt tttgatgagg 18480 gaagaaaactaatgccattt ctgaaacgtt ttttagaatc caggcttctt aatccacaga 18540 agcaaagataaaggaaaaat tacttaatag agtctatatt atgtgaagtc tttttttaac 18600 caaagtttataactattttt atttacaact agaaatgaaa ttaaactgct aaataggcat 18660 tcagattaaataagagtact tcttgcctga gatttatgaa acatttgttt attaaggacc 18720 ccgagactatcttctagata tttgtggagt ggtttaaatg tggtcctgct ttagaacaac 18780 cagaacaaaaacaataggtt gattgataga ttctggtaat tttatgaaat gtaccattta 18840 gttccaaggccagtatttat tatacttcct cctcctcctt ccccaggaaa aattggacca 18900 cctaaactggatatcagaaa ggaggagaag caaatcatga ttgacatatt tcacccttca 18960 gtttttgtaaatggagacga gcaggaagtc gattatgatc ccgaaactac ctgttacatt 19020 agggtgtacaatgtgtatgt gagaatgaac ggaagtgagg tatgtgtttc acatttttca 19080 taatggaaattcttgtgtag ctagcaaaag ttgttccttt ctgtagtgta atgaaaatag 19140 gatgcttataaatattcaag caagactcac agatcataga tttgataaga aaaatatgtg 19200 aatgctattaaagcaaaaat gatacagagt tagtcactaa cactgaccat gtgaatacat 19260 ttagtttttatttgcttcat ttagcagaat ggctctaatc taggattttt cccagtacac 19320 ctccattgctccactggtaa aatgaggatt ttcttcctca ttggcggtga cgtaaagtaa 19380 tattaatacttctgtttccc accagctaaa tttctggaca aatgaatagg acaaatgaac 19440 atttgttagtttaacaacta acaataaaca ttcctgcaat ttgagttttt aaatttacca 19500 tttttataaggaatataaca gatgcatagt atcgtgctgt gttgaagtaa caaattgact 19560 tattgatagtaaagctattt ttagacatgt tcaagtcaat catatatatt tcttcagttg 19620 tttgaccaggactaatatgg tgattttttt tttttttcag attaaaagaa gctgtgcatt 19680 ttcactgttttcttttttca tctagatcca gtataaaata ctcacgcaga aggaagatga 19740 ttgtgacgagattcagtgcc agttagcgat tccagtatcc tcactgaatt ctcagtactg 19800 tgtttcagcagaaggagtct tacatgtgtg gggtgttaca actgaaaagt caaaagaagt 19860 ttgtattaccattttcaata gcagtataaa aggtaagttc ttgccatttt ttttctaaat 19920 atagagggagcagtaactaa aataggatca tgtgaggaaa gcaaactcat ttgcagtttc 19980 aaaagatctgttttaacaat attcatttgc attagttcca ttccttagag aagttcaaaa 20040 ttaaaaataaagcagtttct cacacattta aagatataga acaactaatt tggaaagcca 20100 tgtgatggccccttgaccca ggaaagaaaa aatatataat aagtacataa aactacacaa 20160 atgtttgcatttcaggatta tgaattataa gtcagtttgt tacttcacca tagcaagatg 20220 ttgtgaatccactaaaatgt tctttatggg gattgtaatt taaagactga agagcaggaa 20280 cgatcactggttacaactgt atgacgtgga ctggacaaag actcgagatt aatatccaaa 20340 aaagtttatagatgggtttg aataatagat aatgggtcat ttttataaca ttttacaata 20400 attaattttatttcatcttt taatgtgggg ggtggggagg tagagatata tatgctagaa 20460 cttgaactttagaactgtta ctctcaggtg tcaggaacct taaagaagtt tggacttttc 20520 tttgcagttaggaaatgtaa gtgttcatta tagataattt ataaaatatg aaaaaccaga 20580 agaaaacaaaataatctgta gttctaccat gcaaaaatac cccattattc acagtttgat 20640 attttctttagtttttctac atagacatga tcatattgta tagtatatat gattttgtgt 20700 attgctttaagcatatattg taactcatgc tttgatgatt atctttgtgt gtgtgtgtgt 20760 gtgtgtgtgtgtgtgtgtgt gtgtgtgtat acatatattt ttttccttag gattttttta 20820 gaatagatttcataggttta aaattttatg atgcaagtat attttcgagg aatggaaata 20880 taggctgaagagccagaaca aacacctaaa attcaatact aataacttca agccattata 20940 tgaaggtttcaaataggtga tttaaagcag tgcaatgcat tttatgctaa ttttcgttat 21000 gctatttgaaaaataaaacc cttcttaatg ctcaaaattt gaaataatgc aaatgccgca 21060 gatttgtttttagcctgaat tatacactgc actgatagta tggccacacg gtggcattgc 21120 cgtggctcctgagcagcagc cctgcctgct tgcctcaatg ccttgcagcc agcacttgct 21180 cagcccgtgggacttctaac agcactaatg ttggttttgt agttattttc ctttttactt 21240 ttgtaatgatttaacttttt tcacaccctg taataagcgt aatagaagca ttcattagta 21300 acttccatagttagaaaatt ctgggtaaca aaatgcccta aaggatgccg gacattgtca 21360 cagtatgttagagtgctgtt aatttggaag agagaactat gaaaaaaaaa aaatactgaa 21420 gattgtgctgcaaaaataaa aggcagcatt acagcaaagt acacaatggt gattctagat 21480 gtatgtgcttggaaacaaat ctttcctatt ttatattatt tatgtctggt ttataactaa 21540 ggggactttattcctgtgca gcattgctgt gtgcatgtac atctgtgaag ctttataagt 21600 atttgcagtcgtgaaataaa tgtaacagga aattctgagg agtggggcta aatcatctta 21660 agagcaataaacattgccag aatttctttc tttttttttt tgttgttttg ttttgttttg 21720 aaatggagtctcgctctgtc accaggctgg agtccagtgg cacaatctca gctcactgct 21780 acctccacttcccaggtcca agcgattctc ctgcctcagc ctcccacgta gctgggacta 21840 caggcgcatgccaccacacc cagctaattt ttgtattttt agtagagatg gggtttcacc 21900 atgttggccaagattgtctc gatctcttga cctcgtgatc cgcccacctc gacttcccaa 21960 agtgctgggattacaggctt gagccactgt gcccagctgc cagaatttgt ttctaaggag 22020 aggtatttttaaaattattc ttttgcattt ttaaccgaaa gaaatgcata accctggaaa 22080 cacactgtatgtagggctgt aaggaaatta ttgtagaaaa gctattttaa ctatgttgtg 22140 tcatagtagaatagtcccag aaagttctag aattgcagag ctgggaagaa ccatattgct 22200 atctaagacagatacctaat tttttactca gatttcctaa cttctgcttc cttcagtgtt 22260 ctttaaaactctggccctat tcactaattt gtaaatctat tcaagaatgg cactaagact 22320 ttttgatagataatagcagt attccatctt aattgtaact tgtgatttct gcctttttta 22380 aggttctctttggattccag ttgttgctgc tttactactc tttctagtgc ttagcctggt 22440 attcatctgtttttatatta agaaaattaa tccattgaag gaaaaaagca taatattacc 22500 caagtccttggtaatgtatt taattttttt atatacacta aaaagttaac ttgaaccttt 22560 tttgatagtttcttaaatta tgtttttaaa gtagaatgat attcaacaag atgtcacctg 22620 ccatcaatcagtcagtctac cacacatgca aaatcacata ctgtgtcttt tgctgtgcag 22680 caagtattttgttttttttt ttattctcag ggaaaaacac ggtgagacaa ttctgttaaa 22740 gatcttaaagtctaggggga agaaaagata aatatacatt ggctatataa cgtgtgatag 22800 attcaaagatttatacctga gcccatgagg actttgtcca gctttggggt ggggttagtc 22860 atggcttttccagaggcaat gcctgagcag tcttgaggga catacaggag tttaggcact 22920 aggggacagaatgtgctgag gtccagagcc aagaaagcat gactttgaaa gctctgcaaa 22980 tacagaaacacttcattatg gttggagtat gaaaggaaag ttggccaaaa atgagactaa 23040 cgtaggagtttgacgatttt aatgtccaac atttcaaagc ctttgatgta aagaacattt 23100 aaaggtaacagaatagtatc tatttgagtc aaatatgaca ggaataatat ctgtgatgct 23160 caaagggcacattaaaattt gctataaact acctttaaag tggataagaa ctctcaacag 23220 ttagtagtcaactcacaaat gaattataat attttattct tactagtaat taagcacaaa 23280 gtaaccttgcagtatgccat tttacacgcg ctagagaaga aaaaagaaac ctggatatcc 23340 agtagtaccagcagtagcat aacgggtaaa tgctggtgaa aagtaatcta gccctggtaa 23400 gaataaaagcacacagtggg tattcagtgt aatagtgacg taaacatacc atttctttga 23460 tatggtcactccactcttaa aaaatgtatt ataaggaaat aatttcataa ataaaaagat 23520 gtacaggatatttaacatgt tccatagcta gtaaaaattg gaaacatttc cccatctatt 23580 aaattatttatcagtataaa gaaataatat gagctactaa aaattataat gatgaaggca 23640 agtaatgagaatattactta agtagatgtg cccactttgt aaacataatg tgcgtacatg 23700 tggaagataattgtattagg attctctaga gggacagaac taatggaata tatatataaa 23760 gatatatatataataccaaa taaactcccc tttatataaa tctatatttt atatattata 23820 tattatatatattttatatg ttatataata tatattatat attatatata atatattata 23880 aattatatattttatataat atattataaa ttatatattt tatataatat aagttatata 23940 ttttatatattatattataa attatatgtt tatataatat attataaatt atatatttta 24000 tgtaatataaattatatttt atataatata ttataaatta taaattatat attatatatt 24060 ataaattatatattatataa tatatattaa ttatattttt catatatata tatataaagg 24120 ggagtttatttagtattatt tagtattaac tcacacgatc agaggtccca caataggcca 24180 tctgcaggctgaggagcaag gagagccagt ccaagctcca aacttgaaga attcagagtc 24240 cgatgttcaagggcaggaag cattcagcat gggagaaaga tgtaggctgg gaggctaggc 24300 cagtctctcttttcacattt ttctgcctgc ttacattcta gccatgctgg cagctgatta 24360 gattgtgcccattcgggtta agggcgggtc ttcctttccc agcccactga ctcaaatgtt 24420 aatctcctttggcagcaccc tcacagacac acccaggatc aatactttgt atccttcagt 24480 ccaatcaagttgacactcag tattaaccat cacagtaacg tacaaaaagc aacatatatt 24540 agtaagatatctgatggctt tttaaaaatt ctaaaacttt gtttttaata ttactatggg 24600 acctttcattaaaaagaaat ggcaacatct gattcaccca ttatcctaaa tgtgccattt 24660 ggtggtccattacttcagac ctttgttttt tttgagggta ggcacttaag cttaacaatt 24720 ttttatctttaatcaatttt tctccccata gatctctgtg gtaagaagtg ctactttaga 24780 gacaaaacctgaatcaaaat atgtatcact catcacgtca taccagccat tttccttaga 24840 aaaggaggtggtctgtgaag agccgttgtc tccagcaaca gttccaggca tgcataccga 24900 agacaatccaggaaaagtgg aacatacaga agaactttct agtataacag aagtggtgac 24960 tactgaagaaaatattcctg acgtggtccc gggcagccat ctgactccaa tagagagaga 25020 gagttcttcacctttaagta gtaaccagtc tgaacctggc agcatcgctt taaactcgta 25080 tcactccagaaattgttctg agagtgatca ctccagaaat ggttttgata ctgattccag 25140 ctgtctggaatcacatagct ccttatctga ctcagaattt cccccaaata ataaaggtga 25200 aataaaaacagaaggacaag agctcataac cgtaataaaa gcccccacct cctttggtta 25260 tgataaaccacatgtgctag tggatctact tgtggatgat agcggtaaag agtccttgat 25320 tggttatagaccaacagaag attccaaaga attttcatga gatcagctaa gttgcaccaa 25380 ctttgaagtctgattttcct ggacagtttt ctgctttaat ttcatgaaaa gattatgatc 25440 tcagaaattgtatcttagtt ggtatcaacc aaatggagtg acttagtgta catgaaagcg 25500 taaagaggatgtgtggcatt ttcacttttg gcttgtaaag tacagacttt tttttttttt 25560 taaacaaaaaaagcattgta acttatgaac ctttacatcc agataggtta ccagtaacgg 25620 aacagtatccagtactcctg gttcctaggt gagcaggtga tgccccaggg acctttgtag 25680 ccacttcactttttttcttt tctctgcctt ggtatagcat atgtttttgt aagtttatgc 25740 atacagtaattttaagtaat ttcagaagaa attctgcaag cttttcaaaa ttggacttaa 25800 aatctaattcaaactaatag aattaatgga atatgtaaat agaaacgtgt atatttttta 25860 tgaaacattacagttagaga tttttaaata aagaatttta aaactcgttt ttgtattcat 25920 ttattcaagcacttaggcag tctttcatag ttaagcagaa tatttttata tccactgctt 25980 gtttccatgcattaatgcta 26000 11 20 DNA Artificial Sequence Antisense Oligonucleotide11 tggcatgatc tggtactccc 20 12 20 DNA Artificial Sequence AntisenseOligonucleotide 12 ccacgtcagg aatattttct 20 13 20 DNA ArtificialSequence Antisense Oligonucleotide 13 tctctctctc tattggagtc 20 14 20 DNAArtificial Sequence Antisense Oligonucleotide 14 aagcgatgct gccaggttca20 15 20 DNA Artificial Sequence Antisense Oligonucleotide 15 gaaaattctttggaatcttc 20 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16agctgatctc atgaaaattc 20 17 20 DNA Artificial Sequence AntisenseOligonucleotide 17 tcataatctt ttcatgaaat 20 18 20 DNA ArtificialSequence Antisense Oligonucleotide 18 tctgagatca taatcttttc 20 19 20 DNAArtificial Sequence Antisense Oligonucleotide 19 cctctttacg ctttcatgta20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 ccctgccccagagatttgtg 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21gcggtgccca tctcagccct 20 22 20 DNA Artificial Sequence AntisenseOligonucleotide 22 cccagatccg cggtgcccat 20 23 20 DNA ArtificialSequence Antisense Oligonucleotide 23 ttagttggtg taggcactga 20 24 20 DNAArtificial Sequence Antisense Oligonucleotide 24 acattagttg gtgtaggcac20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 tgttataggattcaattgta 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26atacgatagg gttcatgtta 20 27 20 DNA Artificial Sequence AntisenseOligonucleotide 27 gtactcccaa tatacgatag 20 28 20 DNA ArtificialSequence Antisense Oligonucleotide 28 ctggtactcc caatatacga 20 29 20 DNAArtificial Sequence Antisense Oligonucleotide 29 gatctggtac tcccaatata20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 ggacctgtggcatgatctgg 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31tggatcacca acatgatcag 20 32 20 DNA Artificial Sequence AntisenseOligonucleotide 32 ccctggcttt aactctgacc 20 33 20 DNA ArtificialSequence Antisense Oligonucleotide 33 ccaaccctgg ctttaactct 20 34 20 DNAArtificial Sequence Antisense Oligonucleotide 34 tgtccaaccc tggctttaac20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 actttgcataggcagattct 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36ctgactttgc ataggcagat 20 37 20 DNA Artificial Sequence AntisenseOligonucleotide 37 tacagcaaat tcttctgact 20 38 20 DNA ArtificialSequence Antisense Oligonucleotide 38 cagtttaggt ggtccaattt 20 39 20 DNAArtificial Sequence Antisense Oligonucleotide 39 ctgatatcca gtttaggtgg20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 catgatttgcttctcctcct 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41gtctccattt acaaaaactg 20 42 20 DNA Artificial Sequence AntisenseOligonucleotide 42 ttcctgctcg tctccattta 20 43 20 DNA ArtificialSequence Antisense Oligonucleotide 43 aatcgacttc ctgctcgtct 20 44 20 DNAArtificial Sequence Antisense Oligonucleotide 44 atgtaacagg tagtttcggg20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 ctcacatacacattgtacac 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46tcattctcac atacacattg 20 47 20 DNA Artificial Sequence AntisenseOligonucleotide 47 ttccgttcat tctcacatac 20 48 20 DNA ArtificialSequence Antisense Oligonucleotide 48 gcgtgagtat tttatactgg 20 49 20 DNAArtificial Sequence Antisense Oligonucleotide 49 ctgcgtgagt attttatact20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 acaatcatcttccttctgcg 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51tcacaatcat cttccttctg 20 52 20 DNA Artificial Sequence AntisenseOligonucleotide 52 cactgaatct cgtcacaatc 20 53 20 DNA ArtificialSequence Antisense Oligonucleotide 53 actggcactg aatctcgtca 20 54 20 DNAArtificial Sequence Antisense Oligonucleotide 54 tactgagaat tcagtgagga20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 cagtactgagaattcagtga 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56aaacacagta ctgagaattc 20 57 20 DNA Artificial Sequence AntisenseOligonucleotide 57 cttctgctga aacacagtac 20 58 20 DNA ArtificialSequence Antisense Oligonucleotide 58 ccccacacat gtaagactcc 20 59 20 DNAArtificial Sequence Antisense Oligonucleotide 59 aacaccccac acatgtaaga20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 cttttatactgctattgaaa 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61gcagcaacaa ctggaatcca 20 62 20 DNA Artificial Sequence AntisenseOligonucleotide 62 gtagtaaagc agcaacaact 20 63 20 DNA ArtificialSequence Antisense Oligonucleotide 63 ctaagcacta gaaagagtag 20 64 20 DNAArtificial Sequence Antisense Oligonucleotide 64 taaagtagca cttcttacca20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 ggttttgtctctaaagtagc 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66atggctggta tgacgtgatg 20 67 20 DNA Artificial Sequence AntisenseOligonucleotide 67 gttgctggag acaacggctc 20 68 20 DNA ArtificialSequence Antisense Oligonucleotide 68 aactgttgct ggagacaacg 20 69 20 DNAArtificial Sequence Antisense Oligonucleotide 69 gttccacttt tcctggattg20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 agttcttctgtatgttccac 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71aaagttcttc tgtatgttcc 20 72 20 DNA Artificial Sequence AntisenseOligonucleotide 72 gatggctgcc cgggaccacg 20 73 20 DNA ArtificialSequence Antisense Oligonucleotide 73 tcagatggct gcccgggacc 20 74 20 DNAArtificial Sequence Antisense Oligonucleotide 74 gaagaactct ctctctctat20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 ctacttaaaggtgaagaact 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76actggttact acttaaaggt 20 77 20 DNA Artificial Sequence AntisenseOligonucleotide 77 gagtgatacg agtttaaagc 20 78 20 DNA ArtificialSequence Antisense Oligonucleotide 78 gagtgatcac tctcagaaca 20 79 20 DNAArtificial Sequence Antisense Oligonucleotide 79 tctggagtga tcactctcag20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 gctatgtgattccagacagc 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81ggaaattctg agtcagataa 20 82 20 DNA Artificial Sequence AntisenseOligonucleotide 82 ttacggttat gagctcttgt 20 83 20 DNA ArtificialSequence Antisense Oligonucleotide 83 ttatcataac caaaggaggt 20 84 20 DNAArtificial Sequence Antisense Oligonucleotide 84 tatcatccac aagtagatcc20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 ccgctatcatccacaagtag 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86ctctttaccg ctatcatcca 20 87 20 DNA Artificial Sequence AntisenseOligonucleotide 87 aaggactctt taccgctatc 20 88 20 DNA ArtificialSequence Antisense Oligonucleotide 88 cttctgttgg tctataacca 20

What is claimed is:
 1. A compound up to 50 nucleobases in lengthcomprising at least an 8-nucleobase portion of SEQ ID NO: 11, 12, 13,16, 17, 18, 22, 24, 25, 28, 29, 30, 41, 44, 45, 48, 53, 55, 57, 59, 61,63, 65, 66, 67, 68, 70, 71, 73, 77, 79, 86 or 87 which inhibits theexpression of Interferon gamma receptor
 1. 2. The compound of claim 1which is an antisense oligonucleotide.
 3. The compound of claim 2wherein the antisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 4. The compound of claim 2 wherein the modifiedinternucleoside linkage is a phosphorothioate linkage.
 5. The compoundof claim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 6. The compound of claim 5 wherein the modifiedsugar moiety is a 2′-O-methoxyethyl sugar moiety.
 7. The compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified nucleobase.
 8. The compound of claim 6 wherein the modifiecnucleobase is a 5-methylcytosine.
 9. The compound of claim 2 wherein theantisense oligonucleotide is a chimeric oligonucleotide.
 10. A method ofinhibiting the expression of Interferon gamma receptor 1 in human cellsor tissues comprising contacting said cells or tissues in vitro with thecompound of claim 1 so that expression of Interferon gamma receptor 1 isinhibited.
 11. A composition comprising the compound of claim 1 and apharmaceutically acceptable carrier or diluent.
 12. The composition ofclaim 11 further comprising a colloidal dispersion system.
 13. Thecomposition of claim 11 wherein the compound is an antisenseoligonucleotide.